Reactions of fulvic acid with metal ions

Fulvic acid is a water-soluble humic material that occurs widely in soils and waters and that tends to form water-soluble and water-insoluble complexes with a variety of metal ions, some of which are toxic. This paper presents information on the conditions under which the different types of FA-metal complexes are formed. The solubility in water, separately and after mixing, of FA (2 to 30 mg/100 ml) and eleven metal ions (Fe(III), Al, Cr(III), Pb, Cu, Hg(II), Zn, Ni, Co, Cd and Mn; 1 × 10^?5 moles of each metal ion) was investigated over the pH range 4 to 9. After mixing, the solubility of the components was significantly affected by pH only when less than 20 mg of FA was present. As the systems became richer in FA (22 to 30 mg), most of the metal ions remained in the aqueous phase, likely due to the formation of FA-metal complexes, inhibiting the formation of metal hydroxides. The order in which the eleven metal ions tended to form water-insoluble FA-metal complexes depended on the pH. At pH 6 it was: Fe = Cr = Al > Pb = Cu > Hg > Zn = Ni = Co = Cd = Mn. This order appeared to correlate with the valence, 1st hydrolysis constants and effective hydrated ionic diameters of the metal ions. In general, FA/metals weight ratios of > 2 favored the formation of water-soluble FA-metal complexes; at lower ratios, water-insoluble complexes, which could accumulate in soils and sediments, were formed.     

Comparison of studies on saccharin and sodium nitrite.

A review of long term animal studies of saccharin and sodium nitrite was undertaken to assess the effect of variability of selected protocol elements on the results obtained. These elements were divided into 4 general categories: design, including selection of test animals, basal diet, dosage form and doses of test substance, route of administration, and duration of exposure; observations, including gross observations during life and at necropsy, clinical tests, and histopathology; performance, including conduct of the test and animal husbandry; and analytical procedures, including chemical and statistical analyses. Because many of the protocol elements are not fully discussed in study reports, it was often impossible to determine what actually had been done. The review of various saccharin studies suggests that bladder tumors resulted following in utero exposure. In utero exposure with sodium nitrite did not appear to cause reticuloendothelial changes. The numerous variations in protocol elements in the nitrite studies precluded identification of a prime element responsible for the variation in reticuloendothelial changes observed. It can be concluded from this review that achievement of reproducibility in long term studies requires minimal variation of protocol elements for the new study.     

Reactions of nucleic acid bases with inorganic soil constituents

The adsorption at pH's 4, 6 and 8 of adenine, guanine, cytosine, thymine and uracil on clays (montmorillonite, illite and kaolinite), Fe- and Al-oxides (goethite, hematite and gibbsite), a soil, and on a laboratory-prepared fulvic acid-montmorillonite complex was investigated. Portions of the clays and soil were saturated with H⁺, Fe³⁺ and Ca²⁺.Quantitatively, the extent of adsorption of nucleic acid bases by the clays was proportional to their exchange capacities, but the nature of the dominant cation had only minor effects. By contrast, the adsorption was strongly affected by pH, tending to decrease with increase in pH. Adsorption on goethite and gibbsite was lower than that on clays, while adsorption of nucleic acid bases on soils was slightly lower than that on oxides. The fulvic acid-montmorillonite complex adsorbed substantial, although smaller amounts of purines and pyrimidines, than did montmorillonite alone. The main adsorption mechanism at pH 4 appeared to be cation exchange whereas at pH 8 complex formation between the nucleic acid bases and cations on inorganic surfaces seemed to occur.The results of this and earlier work show that both inorganic and organic soil constituents adsorb nucleic acid bases. Which adsorption reaction predominates will depend on the clay and organic matter content and on the pH.     

Characterization of the p-coumaric acid decarboxylase from Lactobacillus plantarum CECT 748(T)

It was previously reported that cell cultures from Lactobacillus plantarum CECT 748 (T) were able to decarboxylate phenolic acids, such as p-coumaric, m-coumaric, caffeic, ferulic, gallic, and protocatechuic acid. The p-coumaric acid decarboxylase (PDC) from this strain has been overexpressed and purified. This PDC differs at its C-terminal end when compared to the previously reported PDC from L. plantarum LPCHL2. Because the C-terminal region of PDC is involved in enzymatic activity, especially in substrate activity, it was decided to biochemically characterize the PDC from L. plantarum CECT 748 (T). Contrarily to L. plantarum LPCHL2 PDC, the recombinant PDC from L. plantarum CECT 748 (T) is a heat-labile enzyme, showing optimal activity at 22 degrees C. This PDC is able to decarboxylate exclusively the hydroxycinnamic acids p-coumaric, caffeic, and ferulic acids. Kinetic analysis showed that the enzyme has a 14-fold higher K(M) value for p-coumaric and caffeic acids than for ferulic acid. PDC catalyzes the formation of the corresponding 4-vinyl derivatives (vinylphenol and vinylguaiacol) from p-coumaric and ferulic acids, respectively, which are valuable food additives that have been approved as flavoring agents. The biochemical characteristics showed by L. plantarum PDC should be taken into account for its potential use in the food-processing industry.     

Antioxidant mechanism studies on ferulic acid: identification of oxidative coupling products from methyl ferulate and linoleate

In our studies of the chain-breaking antioxidant mechanism of natural phenols in food components, ferulic acid, a phenolic acid widely distributed in edible plants, especially grain, was investigated. The radical oxidation reaction of a large amount of ethyl linoleate in the presence of the methyl ester of ferulic acid produced four types of peroxides as radical termination products. The isolation and structure determination of the peroxides revealed that they had tricyclic structures which consisted of ethyl linoleate, methyl ferulate, and molecular oxygen. Based on the formation pathway of the products, a radical scavenging reaction occurred at the 3'-position of the ferulate radical with the four types of peroxyl radicals of ethyl linoleate. The produced peroxides subsequently underwent intramolecular Diels-Alder reaction to afford stable tricyclic peroxides.     

Antioxidation mechanism studies of caffeic acid: identification of antioxidation products of methyl caffeate from lipid oxidation

As a part of a research project on the elucidation of the chain-breaking antioxidation mechanism of natural phenols in food components, caffeic acid, a polyphenolic acid widely distributed in edible plants, was investigated. The identification and time course analysis of the antioxidation reaction products from methyl caffeate were carried out in the ethyl linoleate oxidation system. The antioxidation reaction produced a quinone derivative of methyl caffeate as an antioxidation product during the initial stage, which was identified by (13)C NMR. The quinone, however, was not the final product, and a further reaction occurred to produce several new peroxides. The isolation and structure determination of the peroxides revealed that they had tricyclic structures, which consisted of ethyl linoleate, methyl caffeate, and molecular oxygen. On the basis of the formation pathway of these products, an antioxidation reaction mechanism of methyl caffeate, including the redox reaction of the caffeate and Diels-Alder reaction of the produced peroxides, was proposed.     

Differential effects of ferulic acid and p-coumaric acid on S phase distribution and length of S phase in the human colonic cell line Caco-2

Ferulic acid (FA) and para-coumaric acid (p-CA) may mediate the protective effects of whole-grain cereals against colon cancer. Therefore, the effects of FA and p-CA on the metabolic activity, proliferation, cell cycle phase distribution, and kinetics of the colonic endothelial tumor cell line Caco-2 was studied. Both compounds at 1500 microM decreased the number of cells to 43-75% of control after 2-3 days of treatment. Cell cycle phase distribution and cell cycle kinetics were determined by flow cytometric analysis after bromodeoxyuridine labeling. Each compound at 1500 microM decreased the proportion of cells in the G(1) phase and increased the proportion of cells in the S and G(2) phases. Treatment with 1500 microM FA significantly increased the length of the S phase, while p-CA did not. It was concluded that FA and p-CA inhibited cell proliferation by presumably affecting different cell cycle phases, and this warrants further investigations because this inhibition may be one explanation for the diet-related protection against cancer.     

Photodegradation of metolachlor: isolation, identification, and quantification of monochloroacetic acid

The photolysis of metolachlor [2-chloro-N-(2-ethyl-6-methylphenyl)-N-(2-methoxy-1-methylethyl) acetamide] in a sunlight simulator under actinic radiation was investigated. The focus of the study was to determine the extent of monochloroacetic acid (MCA) production. MCA was concentrated and derivatized from photolysate as the n-propyl ester using propanol and sulfuric acid and then identified as the ester using GC/MS and GC/ECD. On the basis of regression analysis, it was shown that the direct photodegradation of approximately 10 microM metolachlor followed pseudo-first-order kinetics with respect to the metolachlor concentration, and the half-life of the herbicide ( approximately 74 h) was independent of the pH of the medium. Photolysis in synthetic field water (SFW) resulted in a significant reduction of photolysis time (t(1/2) approximately 9 h). Direct photolysis experiments indicate a 5.19 +/- 0.81% (n=3) conversion of metolachlor to MCA, while photolysis in synthetic field water and in a Don River water sample resulted in 29.8 +/- 4.6% (n = 3) and 12.6 +/- 4.1% (n = 3) conversion, respectively; MCA was shown to be hydrolytically stable over the time course of the photoreaction. The photodegradation of alachlor, butachlor and a model chloroacetanilide, 2-chloro-N-methylacetanilide, in SFW were also investigated.     

Production and isolation of aflatoxin M1 for toxicological studies

One hundred mg aflatoxin M1 was produced and purified for toxicological studies. Aspergillus flavus NRRL 3251 was cultured on rice to produce aflatoxins B1, B2, M1, and M2, B1 and B2 were separated from M1 and M2 by a normal phase low pressure liquid chromatography (LC) column. M1 was then separated from M2 by a reverse phase low pressure LC column. Recoveries of aflatoxins from the LC columns were about 90%. The purified M1 was confirmed by ultraviolet-visible spectrometry, mass spectrometry, nuclear magnetic resonance spectrometry, optical rotation, and its mutagenicity to Salmonella typhimurium TA98.     

Oxidation of diazinon by aqueous chlorine: kinetics, mechanisms, and product studies

The oxidation kinetics and mechanisms of diazinon, an organophosphorus pesticide, by aqueous chlorine were studied under different conditions. The oxidation is of first order with respect to both diazinon and chlorine. The oxidation rate is found to increase with decreasing pH. The second-order rate constants at pH 9. 5, 10.0, 10.5, and 11.0 are determined to be 1.6, 0.64, 0.43, and 0. 32 M(-)(1) s(-)(1), respectively. Based on the rate constants at different temperatures, the activation energy is calculated to be 30 kJ/mol at pH 10.0 with a chlorine-to-diazinon ratio of 11:1, 33 kJ/mol at pH 11.0 with a 11:1 ratio, and 36 kJ/mol at pH 11.0 with a 5:1 ratio, respectively. Diazoxon is identified as the oxidation product by GC-MS. Ion chromatography analysis shows an increase of sulfate concentration as the reaction proceeds, indicating that sulfur is being oxidized to sulfate. This study indicates that oxidation by aqueous chlorine can significantly affect the fate of diazinon in the environment.     

Gastrointestinal absorption and urinary excretion of trans-cinnamic and p-coumaric acids in rats

trans-Cinnamic acid (CIN) and p-coumaric acid (COU) are ingested by humans in their diet. While the metabolism and health benefits of CIN have been widely documented, little is known about its absorption sites, and there have been few studies dedicated to COU. The gastrointestinal sac technique demonstrated that CIN and COU are absorbed by all digestive organs in rats and partially transported via MCT-mediated carrier. Absorption was lowest in the stomach. Regardless of the organs that were studied, CIN was more efficiently absorbed than COU. After their individual oral administration to rats, CIN and COU were excreted in 0-24 h urine (0.3% and 23% of ingested CIN and COU, respectively). This suggests that COU was less metabolized than CIN. CIN and COU are absorbed across the digestive epithelium and subsequently interact with target tissues. Despite its lower gastrointestinal absorption, COU may have greater health benefits because it seems to be less metabolized than CIN.     

Intestinal absorption of p-coumaric and gallic acids in rats after oral administration

Ferulic acid (FA) and p-coumaric acid (CA) are absorbed by the monocarboxylic acid transporter (MCT) in Caco-2 cells, although gallic acid (GA) is not. Therefore, the MCT is selective for certain phenolic acids. Absorption of orally administered CA and GA in rats was studied to obtain serum pharmacokinetic profiles and to investigate their intestinal absorption characteristics in vivo. Rats were administered 100 micromol/kg body weight of CA and GA, and blood was collected from the portal vein and abdominal artery after administration. CA, GA, and their metabolites were quantified with a highly selective and sensitive coulometric detection method using high-performance liquid chromatography-electrochemical detection. Ingested CA was rapidly absorbed in the gastrointestinal tract in an intact form. The serum concentration of intact CA in the portal vein peaked 10 min after dosing (C(max) was 165.7 micromol/L). In contrast, GA was slowly absorbed, with a t(max) for intact GA of 60 min and a C(max) of 0.71 micromol/L. The area under the curve for intact CA and GA was calculated from the serum concentration profile in the portal vein to be 2991.3 and 42.6 micromol min L(-)(1), respectively. The relative bioavailability of CA against GA was about 70. This is the first demonstration that absorption efficiency of CA is much higher than that of GA in vivo. The absorption characteristics of CA are clearly different from those of GA. These findings are in good agreement with the results obtained in vitro using a Caco-2 cell system.     

Pathways for the hydrolysis of phorate: product studies by (31)P NMR and GC-MS

A new intramolecular mechanism is proposed for the hydrolysis of phorate. (31)P NMR was used to study the formation of P-containing products of phorate hydrolysis in situ. When hydrolysis was followed by (31)P NMR, a dominant P-containing product was found and was identified to be diethyl dithiophosphate using methylation and GC-MS. Combining the data from phorate hydrolysis at three different temperatures, thermodynamic parameters were calculated. The contributions of various possible pathways to phorate hydrolysis are discussed.     

Sugar fragmentation in the maillard reaction cascade: isotope labeling studies on the formation of acetic acid by a hydrolytic beta-dicarbonyl cleavage mechanism

The formation of acetic acid was elucidated based on volatile reaction products and related nonvolatile key intermediates. The origin and yield of acetic acid were determined under well-controlled conditions (90-120 degrees C, pH 6-8). Experiments with various 13C-labeled glucose isotopomers in the presence of glycine revealed all six carbon atoms being incorporated into acetic acid: C-1/C-2 ( approximately 70%), C-3/C-4 ( approximately 10%), and C-5/C-6 (approximately 20%). Acetic acid is a good marker of the 2,3-enolization pathway since it is almost exclusively formed from 1-deoxy-2,3-diulose intermediates. Depending on the pH, the acetic acid conversion yield reached 85 mol % when using 1-deoxy-2,3-hexodiulose (1) as a precursor. Hydrolytic beta-dicarbonyl cleavage of 1-deoxy-2,4-hexodiuloses was shown to be the major pathway leading to acetic acid from glucose without the intermediacy of any oxidizing agents. The presence of key intermediates was corroborated for the first time, i.e., tetroses and 2-hydroxy-3-oxobutanal, a tautomer of 1-hydroxy-2,3-butanedione, also referred to as 1-deoxy-2,3-tetrodiulose. The hydrolytic beta-dicarbonyl cleavage represents a general pathway to organic acids, which corresponds to an acyloin cleavage or a retro-Claisen type reaction. Although alternative mechanisms must exist, the frequently reported hydrolytic alpha-dicarbonyl cleavage of 1 can be ruled out as a pathway forming carboxylic acids.     

Reactions of gases on calcareous stones under dry conditions in field and laboratory studies

Dry deposition of gases plays an important role for the deterioration of stone materials and a better understanding of the processes involved will improve our ability to maintain stone monuments and buildings. As a part of an EU-project an investigation with four calcareous stone types have been exposed outdoor at two test sites in Norway for two years. The exposure has been carried out in sheltered position and the amount of reaction products and the penetration depth of SO₂ into the stones was determined as soluble sulphate after half a year and after one and two years. Even if most of the sulphate was found in the upper 0.3 mm of the stone, there was an increase in the sulphate content in stone even down to the center of the stone sample. In laboratory tests with SO₂, NO₂ and changing relative humidity the synergistic effect of NO₂ and the importance of the relative humidity was investigated. The uptake rates were calculated from the laboratory studies by analyzing the gas concentrations before and after the exposure chamber. By calculating the deposition velocity from the field study by using the amount of sulphate found in the stones together with the average outdoor concentration of SO₂ at the test sites, the values were a magnitude higher than in the laboratory test, highest at the industrial paper mill sites with high concentrations both of SO₂ and some hypochlorite and lower in urban atmosphere with fairly low values of SO₂ and high values of NO₂.